X-ray diffraction data is by far the main source of 3-dimensional structure information at atomic resolution for proteins. While a great deal of information is potentially available from this technique, its application requires the ability to prepare crystals of size and order suitable for X-ray analysis. This has proved especially difficult for membrane proteins. Cytochrome reductase (the cytochrome bc1 complex) is a membrane protein complex which makes up the middle segment of the mitochondrial respiratory chain. The respiratory chain is responsible for biological oxidation and for conservation of the energy released in the form of a proton electrochemical potential gradient across the mitochondrial inner membrane. Energy from this gradient is then used to synthesize ATP or to do work by transporting substances across the membrane. In recent years a number of mitochondrial myopathies have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in cytochrome reductase. The 2 largest subunits of the beef bc1 complex belong to a family of mitochondrial import processing peptidases. In at least the case of the potato tuber enzyme, import processing activity can be demonstrated in the isolated cytochrome reductase complex. Thus this enzyme is highly interesting with respect to mitochondrial import and processing as well as electron transport. We have developed procedures for preparing several different forms of crystals of mitochondrial cytochrome reductase from bovine or pork heart. We have collected data and determined the space group of hexagonal (P61/22) and orthorhombic (C222/1) crystals from beef heart. Both of these forms now diffract to 3.8 Angstroms at best. We propose to (a)determine the structure at intermediate resolution using such crystals, and (b) further improve the crystals to diffract at better than 3.5 Angstroms with good completeness, so we can obtain a density map with high enough resolution to begin chain tracing to determine atomic coordinates. To obtain the intermediate resolution map we will phase reflections using a combination of molecular replacement (from the low resolution structure of the Neurospora complex) and isomorphous replacement (using synthetic inhibitors incorporating heavy atoms to form derivatives). To improve the order of the crystals we will concentrate mainly on improving homogeneity of the enzyme preparation and optimizing the amount of lipid and detergent in the crystals.